Molecular genetics

Question 1

There are 3 types of restriction endonucleases, which type is useful for gene manipulation?

Answer 1

Restriction endonuclease II

Question 2

Which DNA sequence does restriction endonuclease target?

Answer 2

Palindrome

Question 3

Which bond does ligase reform?

Answer 3

Phosphodiester bond

Question 4

How does ligase work?

Answer 4

two sticky ends form hydrogen bond first, then ligase come along, re-forms phosphodiester bond between two sticky ends

Question 5

Role of DNA polymerase I (3)

Answer 5

5’>3’ polymerase, 5’>3’ & 3’>5’exonuclease

Question 6

Role of Klenow Fragment DNA polymerase

Answer 6

5’>3’ polymerase, 3’>5’ exonuclease

Question 7

Application of Klenow fragment DNA polymerase

Answer 7

used in Sanger sequencing, synthesis of second strand of cDNA (cloning)

Question 8

Role of Taq polymerase

Answer 8

5’>3’ polymerase activity only

Question 9

Application of Taq polymerase

Answer 9

in PCR (require primer)

Question 10

What are the 4 general types of DNA polymerase used in lab studies and diagnostics?

Answer 10

1. DNA Polymerase I
2. Klenow Fragment DNA polymerase
3. Reverse Transcriptase
4. Taq polymerase

Question 11

Steps of cloning DNA(3)

Answer 11

1. Cut a fragment of DNA of interest
2. Insert into DNA cloning vector creating recombinant DNA molecule
3. Insert vector into host cell

Question 12

3 features of cloning vector

Answer 12

1. be able to self-replicate with origin of replication
2. Must have multiple cloning site (MCS),where endonuclease can detect
3. Must contain selectable marker such as antibiotic resistance

Question 13

Components required for PCR (7)

Answer 13

1. DNA polymerase
2. Primer
3. dNTPs
4. Magnesium
5. Chloride
6. Buffer
7. DNA

Question 14

What is the role of magnesium and chloride for PCR?

Answer 14

Cofactor required for DNA polymerase activity

Question 15

What is primer used in PCR?

Answer 15

It is 2 short, synthetic oligonucleotides which bind to top and bottom strand of target DNA template.

Question 16

4 Steps of PCR

Answer 16

1. Denaturation
2. Annealing of primer
3. Primer extension
4. Amplification

Question 17

Temperature for denaturation of PCR

Answer 17

95

Question 18

Temperature for annealing of PCR

Answer 18

45-65

Question 19

Temperature for primer extension in PCR

Answer 19

Depending on which polymerase used, but Taq pol : 72

Question 20

How do we amplify mRNA?

Answer 20

Need to convert mRNA to cDNA

Question 21

Amplification of cDNA

Answer 21

1. isolate mRNA from cells by using poly A tail (add oligo-dT primer)
2. Add dNTP and reverse transcriptase to synthesize cDNA.
3. Destroy mRNA (add alkali which cleave mRNA)
4. Add DNA polymerase to generate 2nd strand of DNA by removing RNA and replacing it with DNA.
5. Add DNA ligase to repair cleaved backbone